The present invention relates to the introduction of genes into cells. In particular, the present invention relates to compositions and methods of nucleic acid formulation for gene delivery.
There are a number of techniques for the introduction of genes into cells. One common method involves viruses that have foreign genes (e.g., transgenes) incorporated within the viral DNA. However, the viral genes are also delivered with the desired gene and this can lead to undesirable results.
Nonviral gene delivery systems are being developed to transfect mammalian host cells with foreign genes. In such approaches, nucleic acid is typically complexed with carriers that facilitate the transfer of the DNA across the cell membrane for delivery to the nucleus. The efficiency of gene transfer into cells directly influences the resultant gene expression levels.
The carrier molecules bind and condense DNA into small particles which facilitate DNA entry into cells through endocytosis or pinocytosis. In addition, the carrier molecules act as scaffolding to which ligands may be attached in order to achieve site specific targeting of DNA.
The most commonly used DNA condensing agent for the development of nonviral gene delivery systems is polylysine in the size range of dp 90-450. Its amino groups have been derivatized with transferrin, glycoconjugates, folate, lectins, antibodies or other proteins to provide specificity in cell recognition, without compromising its binding affinity for DNA. However, the high molecular weight and polydispersity of polylysine also contribute to a lack of chemical control in coupling macromolecular ligands which leads to heterogeneity in polylysine-based carrier molecules. This can complicate the formulation of DNA carrier complexes and limits the ability to systematically optimize carrier design to achieve maximal efficiency.
Clearly, there is a need for improved methods of gene delivery. Such methods should be amenable to use with virtually any gene of interest and permit the introduction of genetic material into a variety of cells and tissues.
The present invention relates to the introduction of genes into cells. In particular, the present invention relates to compositions and methods of nucleic acid formulation for gene delivery. The invention contemplates cationic peptides containing aromatic amino acids (i.e., phenylalanine, tyrosine and tryptophan) and in particular, tryptophan-containing peptides that mediate gene transfer by condensing DNA into small particles.
The present invention contemplates methods for introducing nucleic acid into cells (both in vivo and in vitro). In one embodiment, the method comprises a) providing: i) an aromatic amino acidxe2x80x94containing peptide capable of binding to nucleic acid, ii) nucleic acid encoding one or more gene products, and iii) cells capable of receiving said nucleic acid, said cells having cell membranes; b) binding said peptide to said nucleic acid to make a complex; c) introducing said complex to said cells under conditions such that said complex is delivered across said cell membrane.
While it is not intended that the invention be limited by the length of the peptide, it is preferred that the peptides of the present invention are less than forty amino acids in length, more preferably less than thirty amino acids in length, and most preferably, less than twenty amino acids in length.
It is also not intended that the present invention be limited by the precise composition of the peptides. A variety of peptides containing aromatic amino acids are contemplated. In one embodiment, the peptides of the present invention comprise L-lysine (Lys) and tryptophan (Trp). In another embodiment,the peptides of the present invention contain L-lysine (Lys), tryptophan (Trp) and cysteine (Cys). In a preferred embodiment, a peptide is contemplated that demonstrates high activity in mediating gene transfer in cell culture, said peptide having the structure (SEQ ID NO:1): Cys-Trp-(Lys)18. Other peptides (including peptides with two, three and four tryptophan residues) are contemplated.
The present invention also contemplates the use of the peptides of the present invention in receptor-mediated gene transfer (both in vitro and in vivo). In one embodiment, the method comprises linking the DNA to a cationic peptide of the present invention (usually an aromatic amino acid- substituted poly-L-lysine) containing a covalently attached ligand, which is selected to target a specific receptor on the surface of the tissue of interest. The gene is taken up by the tissue, transported to the nucleus of the cell and expressed for varying times.
In one embodiment, the receptor-mediated method of the present invention for delivering an oligonucleotide to cells of an animal, comprises a) providing: i) a target binding moiety capable of binding to a receptor present on the surface of a cell in a tissue of an animal, ii) an aromatic amino acidxe2x80x94substituted polylysine capable of binding to nucleic acid, iii) an oligonucleotide encoding one or more gene products, and iv) a recipient animal having cells, said cells having said receptor; b) conjugating said target binding moiety to said substituted polylysine to form a carrier; c) coupling said carrier with said oligonucleotide to form a pharmaceutical composition; and d) administering said composition to said recipient animal under conditions such that said oligonucleotide is delivered to said cells.
As noted above, the present invention contemplates polylysine peptides containing tryptophan for use in gene delivery. In one embodiment, the synthetic peptides contemplated possess a lysine repeat varying from between 3 and 36 residues and comprise one or more tryptophan and cysteine residues. In a preferred embodiment, the peptide comprises 13-18 lysine residues; such peptides which possess a single tryptophan residue enhances gene transfer to cells in culture by up to three orders of magnitude relative to comparable polylysine peptide lacking a tryptophan.
An understanding of how the peptides of the present invention improve the gene delivery in a superior manner is not required to practice the present invention. Nonetheless, it is believed that the mechanism of peptide mediated gene transfer is related to the efficiency of condensing DNA into small particles. While not limited to any particular theory, it is believed that tryptophan plays a specific role in organizing the DNA binding of cationic peptide to produce small condensates that exhibit enhanced gene transfer efficiency. In this manner, the tryptophan-containing peptides of the present invention represent a new class of low molecular weight condensing agents that may be modified with ligands to produce low molecular weight carriers for site specific gene delivery.
It is not intended that the present invention be limited by the nature of the nucleic acid. The target nucleic acid may be native or synthesized nucleic acid. The nucleic acid may be from a viral, bacterial, animal or plant source.
To facilitate understanding of the invention, a number of terms are defined below.
The term xe2x80x9cgenexe2x80x9d refers to a DNA sequence that comprises control and coding sequences necessary for the production of a polypeptide or precursor thereof. The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity is retained.
The term xe2x80x9cwild-typexe2x80x9d refers to a gene or gene product which has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the xe2x80x9cnormalxe2x80x9d or xe2x80x9cwild-typexe2x80x9d form of the gene. In contrast, the term xe2x80x9cmodifiedxe2x80x9d or xe2x80x9cmutantxe2x80x9d refers to a gene or gene product which displays modifications in sequence and or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.
The term xe2x80x9coligonucleotidexe2x80x9d as used herein is defined as a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, usually more than three (3), and typically more than ten (10) and up to one hundred (100) or more. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof.
Because mononucleotides are reacted to make oligonucleotides in a manner such that the 5xe2x80x2 phosphate of one mononucleotide pentose ring is attached to the 3xe2x80x2 oxygen of its neighbor in one direction via a phosphodiester linkage, an end of an oligonucleotide is referred to as the xe2x80x9c5xe2x80x2 endxe2x80x9d if its 5xe2x80x2 phosphate is not linked to the 3xe2x80x2 oxygen of a mononucleotide pentose ring and as the xe2x80x9c3xe2x80x2 endxe2x80x9d if its 3xe2x80x2 oxygen is not linked to a 5xe2x80x2 phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5xe2x80x2 and 3xe2x80x2 ends.
The term xe2x80x9clabelxe2x80x9d as used herein refers to any atom or molecule which can be used to provide a detectable preferably quantifiable) signal, and which can be attached to a nucleic acid or protein. Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like.
The terms xe2x80x9cnucleic acid substratexe2x80x9d and xe2x80x9cnucleic acid templatexe2x80x9d are used herein interchangeably and refer to a nucleic acid molecule which may comprise single- or double-stranded DNA or RNA.
The term xe2x80x9csubstantially single-strandedxe2x80x9d when used in reference to a nucleic acid substrate means that the substrate molecule exists primarily as a single strand of nucleic acid in contrast to a double-stranded substrate which exists as two strands of nucleic acid which are held together by inter-strand base pairing interactions.
The term xe2x80x9csequence variationxe2x80x9d as used herein refers to differences in nucleic acid sequence between two nucleic acid templates. For example, a wild-type structural gene and a mutant form of this wild-type structural gene may vary in sequence by the presence of single base substitutions and/or deletions or insertions of one or more nucleotides. These two forms of the structural gene are said to vary in sequence from one another. A second mutant form of the structural gene may exist. This second mutant form is said to vary in sequence from both the wild-type gene and the first mutant form of the gene. It is noted, however, that the invention does not require that a comparison be made between one or more forms of a gene to detect sequence variations.
The xe2x80x9ctarget cellsxe2x80x9d may belong to tissues (including organs) of the organism, including cells belonging to (in the case of an animal) its nervous system (e.g., the brain, spinal cord and peripheral nervous cells), the circulatory system (e.g., the heart, vascular tissue and red and white blood cells), the digestive system (e.g., the stomach and intestines), the respiratory system (e.g., the nose and the lungs), the reproductive system, the endocrine system (the liver, spleen, thyroids, paradiyrolds), the skin, the muscles, or the connective tissue.
Alternatively, the cells may be cancer cells derived from any organ or tissue of the target organism, or cells of a parasite or pathogen infecting the organism, or virally infected cells of the organism.
Exogenous DNA has been introduced into hepatocytes by targeting the asialoglycoprotein (ASGP) receptor by means of a ligand-poly-L-lysine bioconjugate. See U.S. Pat. No. 5,166,320, hereby incorporated by reference. Such receptor-mediated approaches can be used in combination with the novel peptides of the present invention.
Exogenous DNA has also been introduced for antisense treatment. See U.S. patent application Ser. No. 08/042,943, filed Apr. 5, 1993 (abandoned) and corresponding PCT Publication No. WO 94/23050, hereby incorporated by reference. Such antisense approaches can be used in combination with the novel peptides of the present invention.